An AccQ•Tag Ultra performance liquid chromatography-electrospray ionization-tandem mass spectrometry (AccQ•Tag -UPLC-ESI-MS/MS) method for fast, reproducible and sensitive amino acid quantitation in biological samples, particularly, the malaria parasite Plasmodium falciparum is presented. The Waters Acquity TQD UPLC/MS system equipped with photodiode array (PDA) detector was used for amino acid separation and detection. The method was developed and validated using amino acid standard mixtures containing acidic, neutral, and basic amino acids. For MS analysis, the optimum cone voltage implemented, based on direct infusion analysis of a few selected AccQ•Tag amino acids with multiple reaction monitoring, varied from 29-39 V, whereas the collision energy varied from 15-35 V. Calibration curves were built using both internal and external standardization. Typically, a linear response for all amino acids was observed at concentrations ranges of 3 × 10−3-25 pmol/μL. For some amino acids, concentration limits of detection were as low as 1.65 fmol. The coefficients of variation for retention times were within the ranges of 0.08-1.08%. *Corresponding Author: Virginia Bioinformatics Institute, Virginia Polytechnic Institute and State University Washington Street, Blacksburg, VA 24061-0477, USA vshulaev@vbi.vt.edu Phone: 540-231-3489 Fax: 540-231-2606. SUPPORTING INFORMATION AVAILABLE Plasmodium Falciparum Culture Protocol. Figure S-1. Schematic reaction of amino acid derivatization with AccQ•Tag reagent. Figure S-2. Typical chromatograms for AccQ•Tag tag derivatized amino acids detected in P. falciparum trophozoites, obtained with UPLC-MS/MS using MRM. Figure S-3. Biological reproducibility for AccQ•Tag derivatized amino acids detected in P. falciparum rings or trophozoites. Table S-1. Retention times and parent ions observed for AccQ•Tag derivatized physiological amino acid standards and isotopically labeled amino acids, analyzed by UPLC-MS. Table S-2. Optimal cone voltage and collision energy for selected AccQ•Tag derivatized amino acid standards analyzed by ESI-MS/MS, using a triple quadrupole mass spectrometer, and, either, low flow (20 μL/min) or combined flow (200 μL/min). Table S-3. Peak area reproducibility for AccQ•Tag labeled amino acid standards analyzed by UPLC-MS/MS using 001 s and 002 s dwell time. Table S-4. Dynamic range, high limits of linearity, low limits of linearity, and detection limits for AccQ•Tag physiological amino acids standards. Table S-5. Technical reproducibility for AccQ•Tag derivatized amino acids detected and measured in P. falciparum ring and trophozoite developmental stages. Table S-6. Biological reproducibility across peak areas measured for AccQ•Tag derivatized isotopically labeled amino acid internal standards spiked into red blood cells. Equation S-1 used to calculate limits of detection. This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author Manuscript Anal Chem. Author manuscript; available in PMC 2011 January 15. Published in final edited form as: Anal Chem. 2010 January 15; 82(2): 548–558. doi:10.1021/ac901790q. N IH PA Athor M anscript N IH PA Athor M anscript N IH PA Athor M anscript The coefficients of variation for amino acid quantitation, determined from triplicate UPLC-MS/MS runs, were below 8% on the average. The developed AccQ•Tag-UPLC-ESI-MS/MS method revealed good technical and biological reproducibility when applied to P. falciparum and human red blood cells samples. This study should provide a valuable insight into the performance of UPLC-ESI-MS/ MS for amino acid quantitation using AccQ•Tag derivatization.